Synthesis and characterization of positively charged tPA as a prodrug using a heparin/protamine-based drug-delivery system

Jun F. Liang, Yong T. Li, Maureen E. Connell, Victor C. Yang

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Positively charged peptides [(Arg)7Cys] were successfully linked to tissue-specific plasminogen activator (tPA) using cross-linking agent N-succinimidyl 3-(2-pyridyldithio) propionate. Specific amidolytic activity of this tPA/(Arg)7Cys (termed modified tPA, mtPA) was 3900 IU/μg as compared to 5800 IU/μg of the parent tPA. Both activation of plasminogen with mtPA (Km = 2.7 mM-1) and tPA (Km = 1.1 mM-1) in a purified system followed Michaelis-Menten kinetics. In addition, (Arg)7Cys modification did not result in significant changes in the fibrin-binding ability of tPA, and mtPA still retained a response to fibrinogen similar to that of the parent tPA. Compared with tPA, mtPA showed much stronger heparin affinity, and the heparin/mtPA complex was stable in human plasma. The activity of mtPA in such a complex was inhibited by heparin, and, unlike tPA, the heparin/mtPA complex did not cause statistically meaningful depletion of plasminogen, fibrinogen, and α2-antiplasmin in plasma. Using the chromogenic and the in vitro clot lysis assay, it was demonstrated that the heparin-induced inhibition of the mtPA activity was easily reversed following the addition of an adequate amount of protamine. To enhance the clot-targeting efficiency of the heparin/mtPA complex further, anti-fibrin immunoglobulin (IgG) was conjugated to heparin via an end-point attachment of heparin to the sugar moieties in the Fc region of the IgG. Results show that the activity of mtPA could also be blocked by the heparin/anti-fibrin IgG conjugate. These findings suggest the applicability of the heparin/protamine delivery system to abort the potential bleeding risks associated with clinical use of tPA.

Original languageEnglish
Pages (from-to)59-67
Number of pages9
JournalAAPS PharmSci
Volume2
Issue number1
DOIs
StatePublished - 2000

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